Vascular stem cells and uses thereof

Abstract

The present invention relates to a method for obtaining a cell population enriched in vascular stem cells. Vascular stem cells can be used for a variety of purposes, especially in cell therapy for the treatment of ischemic diseases, including peripheral ischemia and myocardial infarction.

1 . A method for obtaining a cell population enriched in vascular stem cells comprising: 
 a) providing cells isolated from a mammalian artery;    b) combining said cells provided in step (a) with a labelled vital and/or lipophilic substrate for ATP-Binding Cassette Transporters, under conditions appropriate for uptake of the substrate by said cells;    c) determining the amount of labelled substrate present in each cell; and,    d) isolating the population of nucleated cells which contains the lowest amount of substrate, thereby obtaining said cell population enriched in vascular stem cells.    
   
   
       2 . The method of  claim 1 , wherein said labelled substrate for ATP-Binding Cassette Transporters is a vital fluorescent dye.  
   
   
       3 . The method of  claim 2 , wherein said step c) is carried out by exposing the cells to an excitation wavelength which causes fluorescence of the dye and determining fluorescence emission of each cell.  
   
   
       4 . The method of  claim 3 , wherein said dye is Hoechst 33342 dye, said excitation wavelength is about 351 nm and an emission wavelength from 400 to 700 nm is determined for each cell.  
   
   
       5 . The method of  claim 2 , wherein step c) and d) is carried out by using a FACS device.  
   
   
       6 . The method according to  claim 1 , wherein a dye which allows exclusion of dead cells is added in addition to the labelled substrate.  
   
   
       7 . The method of  claim 6 , wherein said dye allowing exclusion of dead cells is propidium iodide.  
   
   
       8 . The method according to  claim 1 , wherein said cells provided in step (a) are isolated from a human artery.  
   
   
       9 . A method for detecting and/or purifying vascular stem cells from mammalian artery cells, comprising binding said compound specifically to a Bcrp1 transporter.  
   
   
       10 . The method according to  claim 9 , wherein said compound which binds specifically to Bcrp1 transporters is an antibody raised against Bcrp1.  
   
   
       11 . The method according to  claim 10 , wherein said antibody is a polyclonal antibody raised against a Bcrp1 transporter.  
   
   
       12 . The use according to  claim 10 , wherein said antibody is a monoclonal antibody raised against a Bcrp1 transporter.  
   
   
       13 . A method for obtaining a cell population enriched in vascular stem cells, comprising 
 a) providing cells isolated from a mammalian artery; and,    b) selecting cells expressing a Bcrp1 transporter.    
   
   
       14 . The method of  claim 13 , wherein step b) is carried out by FACS sorting.  
   
   
       15 . A cell population enriched in vascular stem cells, characterized in that it is isolated from a mammalian artery and in that it expresses a Bcrp1 transporter.  
   
   
       16 . A cell population enriched in vascular stem cells obtainable by the method of  claim 1 .  
   
   
       17 . A cell population enriched in vascular stem cells and isolated from mammalian artery, comprising between 83.6% to 91.6% Sca-1+ cells, between 49% to 49.8% c-kit+ cells, between 53% to 57.6% Flk-1+ cells, between 8.2% to 9.4% CD34+ cells, between 67.4% to 78.8% Lin− cells and between 39.2% to 43.6% CD45− cells.  
   
   
       18 . A human cell population enriched in vascular stem cells obtainable by the method of  claim 8 .  
   
   
       19 . A method for preparing vascular tissue, comprising culturing a cell population enriched in vascular stem cells according to  claim 15 , in the presence of at least one vasculogenic and/or angiogenic growth factor, under conditions suitable for inducing vascular tissue differentiation, thereby obtaining vascular tissue.  
   
   
       20 . The method according to  claim 19 , further comprising one or more expansion phases after differentiation of said vascular tissue.  
   
   
       21 . The method of  claim 19 , wherein said culture step is carried out in a semi-solid vascularization-promoting medium comprising extracellular matrix components.  
   
   
       22 . The method of  claim 21 , wherein said extracellular matrix component is a Matrigel basement membrane or collagen gel.  
   
   
       23 . The method of  claim 21 , wherein said medium comprises VEGF and one or more compounds selected among the group of fetal bovine serum, hydrocortisone, hFGF-B, R3-IGF-1, ascorbic acid, hEGF, GA-1000 and heparin.  
   
   
       24 . A method for preparing a vascularized regenerated mammalian tissue, comprising: 
 a) providing a cell population enriched in vascular stem cells according to  claim 15;  and    b) contacting said cell population with an isolated mammalian tissue under conditions suitable for differentiation of the vascular stem cells into vascular cells, thereby obtaining vascularized regenerated mammalian tissue.    
   
   
       25 . The method of  claim 24 , wherein isolated mammalian tissue consists of in vitro cell cultures selected among hepatocytes, epidermal and dermal cells, pancreatic, skeletal muscle tissue, smooth muscle tissue, myocardiac tissue, adipous, cartilaginous and bone tissue, and wherein cell culture is co-cultured with said cell population enriched in vascular stem cells.  
   
   
       26 . The method of  claim 25 , wherein said co-culture is carried out on a 3D matrix or on a semi-porous scaffold.  
   
   
       27 . A method for obtaining a cell therapy product comprising 
 a) providing a cell composition selected from the group of:    i. a cell population enriched in vascular stem cells wherein said stem cells are isolated from a mammalian artery and express a Bcrp1 transporter.    ii. a vascular tissue prepared by culturing said cell population in the presence of at least one vasculogenic and/or angiogenic agent under conditions suitable for obtaining tissue.    iii. a vascularized regenerated tissue prepared according to  claim 24 .    b) combining an efficient amount of said cell composition with an appropriate vehicle for in vivo administration in a mammal, thereby obtaining a cell therapy product.    
   
   
       28 . A cell therapy product obtainable by the method of  claim 27 .  
   
   
       29 . The cell therapy product according to  claim 27 , characterized in that it is intended for the treatment, in a mammal, of post-ischemic cardiac failure or cardiovascular disease.  
   
   
       30 . The cell therapy product according to  claim 27 , intended for the treatment of peripheral vascular ischemia.  
   
   
       31 . A method for the treatment in a mammal of post-ischemic cardiac failure or cardiovascular disease comprising administering an effective amount of a cell population according to  claim 15 .  
   
   
       32 . The method according to  claim 31 , wherein said mammal is a human suffering from post-ischemic cardiac failure or cardiovascular disease.  
   
   
       33 . The method according to  claim 31 , wherein said cell population enriched in vascular stem cells is isolated from an artery of said human suffering from post-ischemic cardiac failure or cardiovascular disease.  
   
   
       34 . A method for the in vitro screening of angiogenic or anti-angiogenic compounds screening with a cell population according to  claim 15 .  
   
   
       35 . An in vitro method for screening for compounds having angiogenic or anti-angiogenic compound, said method comprising: 
 a) providing a cell population enriched in vascular stem cells according to  claim 15;     b) combining said cell population with a candidate compound;    c) culturing said cell population under conditions appropriate for growth and differentiation into vascular cells; and    d) determining whether the differentiation of vascular stem cells into vascular cell is enhanced or inhibited, thereby indicating that said candidate compound is respectively an angiogenic or an anti-angiogenic compound respectively.    
   
   
       36 . A composition comprising; 
 a) a cell population enriched in vascular stem cells according to  claim 15;  and    b) a physiologically acceptable carrier.    
   
   
       37 . A kit comprising: 
 a), a cell population enriched in vascular stem cells according to  claim 15;  and    b) reagents necessary for culturing said vascular stem cells.    
   
   
       38 . The kit according to  claim 37 , further comprising an extracellular matrix.  
   
   
       39 . The kit according to  claim 37 , wherein said extracellular matrix is a Matrigel basement membrane or collagen gel.